This is a K08 application from Jae-Woo Lee, MD, an anesthesiologist at the University of California, San Francisco, who is establishing himself as an investigator in the use of cell-based therapy with mesenchymal stem cells (MSC) for treatment of acute lung injury. This application will provide Dr. Lee with the support necessary to accomplish the following goals: (1) develop two innovative and synergistic human acute lung injury models; (2) obtain expertise with molecular biology methods to study the mechanism underlying the therapeutic effect of MSC; and (3) develop cell-based therapy with MSC transfected with plasmids overexpressing key soluble factors. To achieve these goals, Dr. Lee has assembled a Scientific Advisory Committee chaired by Dr. Michael A. Matthay, a renowned intensivist and expert in acute lung injury within the Cardiovascular Research Institute at UCSF. By the end of the grant, Dr. Lee will become an independent investigator focused on translational critical care research in the area of acute lung injury. In this application, Dr. Lee will test the hypothesis that allogeneic human mesenchymal stem cells will restore alveolar fluid clearance and maintain lung endothelial permeability by modulating the proinflammatory response and by up-regulating the release of protective growth factors and anti-inflammatory cytokines in two innovative models of human acute lung injury. MSC, due to its pluripotent nature and its ability to secrete multiple paracrine factors such as growth factors (KGF, HGF) and anti-inflammatory cytokines (IL-10, IL-1ra) can treat multiple abnormalities simultaneously. In Aim 1, he will determine the mechanistic effects of human MSC on alveolar epithelial fluid transport in an in vitro model of human alveolar epithelial type II cells grown in Transwell plates with an air-liquid interface that are injured by cytomix, a mixture of the most biologically active cytokines found in ALI pulmonary edema fluid (IL-1f3, TNFa and IFNv). In Aim 2, he will test the therapeutic effect of intrapulmonary instillation of human MSC on alveolar epithelial fluid clearance and lung endothelial permeability after the establishment of endotoxin injury in an ex vivo perfused human lung preparation. In Aim 3, he will determine if transfection of allogeneic human MSC with plasmids over-expressing key soluble factors will lead to further protection in terms of alveolar fluid clearance and lung endothelial and epithelial permeability to protein in both human lung injury models. The support from this K08 grant will provide Dr. Lee with the necessary skills to become a successful independent investigator. Public Health Relevance: Despite extensive research into the pathophysiology, mortality from acute respiratory distress syndrome remains at 40%. Current treatment options remain primarily supportive with lung protective ventilation and fluid conservative strategy. Innovative therapies are needed.